The effects of extracted and purified anthocyanin (ACY) from mature berries of Vaccinium myrtillus L., on growth parameters, antioxidant enzymes, total phenolic (TP) content, total flavonoid (TF) content, and 2,2diphenyl-l-picrylhydrazyl (DPPH) radical scavenging activity were investigated in the roots of barley (Hordeum vulgare L.) cultivars Akhisar-98 and Ergine1-90 under ionizing irradiation (IR; 10 Gy). The responses to IR and ACY-rich extract treatment in the two cultivars differed; Akhisar-98 was more radiosensitive than Ergine1-90 in terms of root development, but IR-induced decreases in Akhisar-98 root length were alleviated by ACY-rich extract (100 uM). Peroxidase (POD) activity in Akhisar and Erginel cultivars decreased, while superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) activities increased. The ACY-rich extract increased POD activity, and decreased SOD, GR, and APX activities in the two barley cultivars. Although TP content increased in the Erginel cultivar more than the Akhisar cultivar, TF contents did not differ between the control and irradiated roots of the Erginel cultivar. Since TP content, TF content, and DPPH radical scavenging activity increased and H202 content decreased after ACYrich extract treatment, ACY-rich extract treatment enhanced antioxidant activity, especially in the Akhisar cultivar. Treatment with ACY-rich extract conferred a greater tolerance to IR stress, especially after irradiation, in the two barley cultivars exposed to IR and exogenic ACYrich extract. These findings indicate that the Erginel cultivar is more IR-tolerant than the Akhisar cultivar and that exogenous ACY-rich extract treatment promotes antioxidant capacity in barley cultivars under IR stress.